Stimulation of endothelial cells by pro-angiogenic signals released in the chronic inflammatory milieu initiates cellular events required for morphological change, migration and proliferation. Rho-family GTPases (Pho, Rac and Cdc42) regulate the molecular events which mediate these motile responses in other cells. This project will investigate the role of one such intracellular pathway, the Rac/Cdc42-mediated activation of a target kinase Pak (p21-activated kinase), in regulating the cytoskeletal changes required for endothelial cell motility. Initially we will investigate whether pro-inflammatory/pro-angiogenic stimuli result in activation of endogenous Pak kinase activity or change in intracellular localization of Pak in endothelial cells. The second aim will investigate whether Pak can regulate the specific morphological changes required to initiate motility in stimulated endothelial cells. This section will utilize a wide variety of specific Pak mutants developed to successfully address this question in other cell types. Domains in Pak., important in the regulation of endothelial cell morphology. To investigate the mechanism by which Pak regulates the cytoskeleton in stimulated endothelial cells, the third aim will characterize PIX, a putative guanine nucleotide exchange factor, which binds tightly to Pak and may interact with pak in mediating cytoskeletal change. Using these molecular and biochemical approaches we will probe the mechanism by which rho-family GTPases and their downstream targets are involved in the signal transduction pathways associated with increased angiogenesis during chronic inflammation.